The present invention relates to methods and compositions for the inhibition of cell signal transduction associated with cell proliferative disorders. In particular, the invention relates to particular indolylquinone compounds that inhibit protein tyrosine kinase/adaptor protein interactions, and methods for utilizing such compounds. The present invention also relates to methods for treating insulin-related disorders using certain indolylquinone compounds. In particular, the invention is directed to methods for activating the insulin receptor tyrosine kinase in an animal.
2.1 INDOLYLQUINONES
Research interest concerning indolylquinones grew out of early observations that extracts of Chaetomium exhibited antibiotic properties. These observations led researchers to attempt the isolation of active species from cultures of these microorganisms. For example, Brewer et al. disclose the isolation of a purple pigment, which was termed cochliodinol, from isolates of Chaetomium cochliodes and Chaetomium globosum (1968, xe2x80x9cThe Production of Cochliodinol and a Related Metabolite by Chaetomium Species,xe2x80x9d Can. J. Microbiol. 14:861-866). Brewer et al. also disclose the synthetic conversion of cochliodinol to a diacetate compound. Id. Further, the antifungal properties of cochliodinol have also been documented (Meiler et al., 1971, xe2x80x9cThe Effect of Cochliodinol, a Metabolite of Chaetomium cochliodes on the Respiration of Microspores of Fusarium oxysporum,xe2x80x9d Can. J. Microbiol. 17: 83-86).
The structure of cochliodinol was elucidated by Jerram et al. in 1975. (1975, xe2x80x9cThe Chemistry of Cochliodinol, a Metabolite of Chaetomium spp.,xe2x80x9d Can. J. Chem. 53:727-737). Jerram et al. reported the structure of cochliodinol as: 2,5-dihydroxy-3,6-di(5xe2x80x2-(2xe2x80x3-methylbut-xcex942xe2x80x3-ene)-indolyl-3xe2x80x2)-cyclohexadiene-1,4-dione. The conversion of cochliodinol to various other derivatives, including its dimethyl and diacetyl analogues, was also disclosed. Id. Some of these derivatives were highly colored and suitable for use as dyes, while others were colorless. Id. Sekita discloses the isolation of other bis(3-indolyl)-dihydroxybenzoquinones, including isocochliodinol and neocochliodinol from Chaetomium muroum and C. amygdalisporum (1983, xe2x80x9cIsocochliodinol and Neocochliodinol, Bis(indolyl)-benzoquinones from Chaetomium spp.,xe2x80x9d Chem. Pharm. Bull. 31(9): 2998-3001).
Despite the therapeutic potential of cochliodinol and its derivatives, efficient methods suitable for large scale production of these compounds have remained elusive. U.S. Pat. No. 3,917,820 to Brewer et al. discloses the purple pigment cochliodinol and a process for its production by culturing various types of Chaetomium under aerobic conditions. However, the methods of Brewer require long incubation periods for cochliodinol production (2-8 days), the use of benzene, a known carcinogen, to effect chromatographic separation of cochliodinol from the culture and are limited to the few naturally occurring compounds. Moreover, Brewer discloses the isolation of only small quantities (0.75 grams) of cochliodinol from Chaetomium.
Another class of indolylquinones known as the asterriquinones in which the nitrogen of the indole ring is substituted, has been shown to exhibit antitumor activity. Arai et al. proposed the general name xe2x80x9casterriquinonesxe2x80x9d for the class of indolylquinones based upon asterriquinone (1981, xe2x80x9cMetabolic Products of Aspergillus terreus IV. Metabolites of the Strain IFO 8835. (2) The Isolation and Chemical Structure of Indolyl Benzoquinone Pigments,xe2x80x9d Chem. Pharm. Bull. 29(4): 961-969). It should be noted that as used herein, the term xe2x80x9casterriquinonexe2x80x9d has a more general meaning, and is used interchangeably with the term xe2x80x9cindolylquinone.xe2x80x9d Yamamoto et al. disclose the antitumor activity of asterriquinone, i.e., 2,5-bis[N-(1xe2x80x3,1xe2x80x3-dimethyl-2xe2x80x3-propenyl)indol-3xe2x80x2-yl]-3,6-dihydroxy-1,4-benzoquinone, and its isolation from the fungus Aspergillus terreus (1976, xe2x80x9cAntitumor Activity of Asterriquinone, a Metabolic Product from Aspergillus terreus,xe2x80x9d Gann 67:623-624).
Arai et al. disclose the isolation and characterization of 11 different kinds of bisindolyl-dimethoxyl-p-benzoquinones from Aspergillus terreus. Id. The isolation and structural determination of a number of other asterriquinones have also been reported. (Arai et al. 1981, xe2x80x9cMetabolic Products of Aspergillus terreus VI. Metabolites of the Strain IFO 8835. (3) the Isolation and Chemical Structures of Colorless Metabolites,xe2x80x9d Chem. Pharm. Bull. 29(4): 1005-1012; Kaji et al., 1994, xe2x80x9cFour New Metabolites of Aspergillus Terreusxe2x80x9d, Chem. Pharm. Bull. 42(8): 1682-1684). However, the separation of asterriquinones is troublesome because there are so many kinds of homologous pigments in the Aspergillus extracts. Moreover, the chromatographic purification of asterriquinones is typically carried out using benzene, a known carcinogen, as a solvent. Finally, only milligram quantities of asterriquinones have actually been isolated from these natural sources.
In view of their potential as anticancer agents, research has been directed to determination of the relationship between structure and antitumor activity of asterriquinones. For example, Arai et al. reported a study in which hydroxyl benzoquinone derivatives obtained by demethylation of bisindolyl-dimethoxyl-p-benzoquinones were found to have greater antitumor activity than the methoxyl derivatives (1981, xe2x80x9cMetabolic Products of Aspergillus terreus V. Demethylation of Asterriquinones,xe2x80x9d Chem. Pharm. Bull. 29(4): 991-999). Shimizu et al. noted that the presence of free hydroxyl groups in the benzoquinone moiety, as well the number and position of tert-, isopentenyl, or both pentyl groups, seems to have an effect on the antitumor activity of the compound (1982, xe2x80x9cAntitumor Effect and Structure-Activity Relationship of Asterriquinone Analogs,xe2x80x9d Gann 73: 642-648). In an attempt to obtain information towards the development of more potent asterriquinone derivatives, Shimizu et al. conducted an investigation into the structure-activity relationship of asterriquinones in which the action mechanism of asterriquinone in its antitumor activity with reference to its interaction with DNA molecules and the plasma membrane of tumor cells was studied (1990, xe2x80x9cInteraction of Asterriquinone with Deoxyribonucleic Acid in Vitro,xe2x80x9d Chem. Pharm. Bull. 38(9): 2617-2619). It was reported that a correlation exists between the pKa value of the asterriquinone derivative and its antitumor activity. Id. Maximum antitumor activity was observed for compounds with pKa""s in the range of 6-7. Id.
Analysis of structure-activity relationships has led to attempts to obtain compounds with more potent antitumor activity by chemical modification of asterriquinone and related compounds isolated from natural sources (Shimizu et al., 1982, xe2x80x9cAntitumor Activity of Asterriquinones from Aspergillus Fungil IV. An Attempt to Modify the Structure of Asterriquinones to Increase the Activity,xe2x80x9d Chem. Pharm. Bull. 30(5): 1896-1899). Although benzoquinone derivatives having aziridinyl groups in the molecule such as mitomycin C, carbazilquinone or xe2x80x9cE 39xe2x80x9d are well known potent anticancer agents, replacement of the functional groups at the 3 and 6 positions in the benzoquinone moiety of asterriquinone failed to enhance its antitumor potency. Id. Similarly, the introduction of an ethyleneimino group into the molecule did not increase antitumor activity. A dimethylallyl derivative of asterriquinone showed moderate activity against the ascites and solid tumors of Ehrlich carcinoma, while an allyl derivative did not. It was suggested that in order to enhance the antitumor activity, it may be necessary not only to alter the pKa value by alkylation, but also to introduce hydrophilic groups into the molecule.
In addition to their demonstrated antitumor activity, asterriquinone and some of its analogues have also been shown to be strong inhibitors of HIV-reverse transcriptase (Ono et al., 1991, xe2x80x9cInhibition of HIV-Reverse Transcriptase Activity by Asterriquinone and its Analogues,xe2x80x9d Biochem. Biophys. Res. Commun. 174(1): 56-62).
2.2 PROTEIN PHOSPHORYLATION AND SIGNAL TRANSDUCTION
Cells receive signals from their environment through the binding of extracellular molecules to the cell surface. These extracellular signals are essential for the correct regulation of such diverse cellular processes as proliferation, differentiation, chemotaxis, contractility, secretion, contact inhibition, cell survival, neurite outgrowth and metabolism, in particular, the metabolism of glucose. The extracellular molecules, which can be, inter alia, hormones such as insulin, growth factors, lymphokines, or neurotransmitters, are ligands that bind to the extracellular domains of specific cell surface receptors. The binding of these ligands to their receptors triggers cascades of reactions that bring about both the amplification of the original stimulus and the coordinate regulation of cellular processes. In addition to normal cellular processes, receptors and their extracellular ligands may be involved in abnormal or potentially deleterious processes such as virus-receptor interaction, inflammation and cellular transformation to cancerous states. In addition, it is believed that impaired insulin-stimulated glucose uptake observed in diabetic patients may be associated with altered insulin receptor signal transduction. Goodyear et al., 1995, J. Clin. Invest. 95:2195-2204.
A central feature of this signaling process, known as signal transduction, is the reversible phosphorylation of certain proteins. The phosphorylation or dephosphorylation of amino acid residues triggers conformational changes in regulated proteins that alter their biological properties. Proteins are phosphorylated by protein kinases and are dephosphorylated by protein phosphatases. Protein kinases and phosphatases are classified according to the amino acid residues they act on, with one class being serine-threonine kinases and phosphatases (reviewed in Scott, J. D. and Soderling, T. R., 1992, 2:289-295), which act on serine and threonine residues, and the other class being the tyrosine kinases and phosphatases (reviewed in Fischer, E. H. et al., 1991, Science 253:401-406; chlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212), which act on tyrosine residues. The protein kinases and phosphatases may be further defined as being receptors, i.e., the enzymes are an integral part of a transmembrane, ligand-binding molecule, or as non-receptors, meaning they respond to an extracellular molecule indirectly by being acted upon by a ligand-bound receptor. Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the relative activities, at that instant, of the protein kinases and phosphatases that catalyze these reactions.
The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Cantley, L. C. et al., 1991, Cell 64:281-302; Hunter, T., 1991, Cell 64:249-270; Nurse, 1990, Nature 344:503-508; Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212). Subversion of normal growth control pathways leading to oncogenesis has been shown to be caused by activation or over-expression of protein tyrosine kinases which constitute a large group of dominant oncogenic proteins (reviewed in Hunter, T., 1991, Cell 64:249-270).
2.3 PROTEIN TYROSINE KINASES
2.3.1. Receptor-Type Protein Tyrosine Kinases
Many cellular functions are mediated by the binding of growth factor ligands to membrane-bound protein tyrosine kinase (xe2x80x9cPTKxe2x80x9d) receptors. Receptor-type protein tyrosine kinases having transmembrane topology have been studied extensively. The binding of certain ligands to the extracellular domain of a receptor protein tyrosine kinase is thought to induce dimerization of the receptor, resulting in the reversible auto-phosphorylation of receptor tyrosine residues within the intracellular domain of the tyrosine kinase. These individual phosphotyrosine residues may then serve as specific binding sites for a host of cytoplasmic signaling molecules, thereby activating various signal transduction pathways (Ullrich A., and Schlessinger, J., 1990, Cell 61:203-212).
The mechanism by which insulin receptor transmits signals to the interior of the cell upon insulin binding it slightly different. The insulin receptor is a disulfide-linked heterotetramer (xcex12xcex22). Therefore, it does not dimerize upon insulin binding. Rather, insulin interaction with the extracellular portion of the insulin receptor causes a conformational change in the receptor that in turn causes the intracellular tyrosine kinases to become phosphoylated. As in the case with PTK receptors that dimerize, the individual phosphotyrosine residues may serve as binding sites for other molecules in the insulin signaling cascade.
2.3.2. Non-Receptor-Type Protein Tyrosine Kinases
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases, may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrane domain. Within this broad classification, one can divide the known cytoplasmic protein tyrosine kinases into eleven distinct morphotypes, including the SRC family, the FES family, the ABL family, the Zap 70 family and the JAK family. While distinct in their overall molecular structure, members of these morphotypic families of cytoplasmic protein tyrosine kinases may share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 and SH3 domains. These non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson, T. and Gish, G., 1992, Cell 71:359-362).
While the metabolic roles of cytoplasmic protein tyrosine kinases are less well understood than that of the receptor-type protein tyrosine kinases, significant progress has been made in elucidating some of the processes in which this class of molecules is involved. For example, members of the src family, lck and fyn, have been shown to interact with CD4/CD8 and the T cell receptor complex, and are thus implicated in T cell activation, (Veillette, A. and Davidson, D., 1992, TIG 8:61-66), certain cytoplasmic protein tyrosine kinases have been linked to certain phases of the cell cycle (Morgan, D. O. et al., 1989, Cell 57: 775-786; Kipreos, E. T. et al., 1990, Science 248: 217-220; Weaver et al., 1991, Mol. Cell. Biol. 11:4415-4422), and cytoplasmic protein tyrosine kinases have been implicated in neuronal development (Maness, P., 1992, Dev. Neurosci 14:257-270). Deregulation of kinase activity through mutation or overexpression is a well-established mechanism underlying cell transformation (Hunter et al., 1985, supra; Ullrich et al., supra).
2.4 ADAPTOR PROTEINS
Adaptor proteins are intracellular proteins having characteristic conserved peptide domains (SH2 and/or SH3 domains, as described below) which are critical to the signal transduction pathway. Such adaptor proteins serve to link protein tyrosine kinases, especially receptor-type protein tyrosine kinases to downstream intracellular signaling pathways such as the RAS signaling pathway. It is thought that such adaptor proteins may be involved in targeting signal transduction proteins to the correct site in the plasma membrane or subcellular compartments, and may also be involved in the regulation of protein movement within the cell.
Such adaptor proteins are among the protein substrates of the receptor-type protein tyrosine kinases, and have in common one or two copies of an approximately 100 amino acid long motif. Because this motif was originally identified in c-Src-like cytoplasmic, non-receptor tyrosine kinases it is referred to as a Src homology 2 (SH2) domain. SH2-containing polypeptides may otherwise, however, be structurally and functionally distinct from one another (Koch, C. A. et al., 1991, Science 252:668-674). SH2 domains directly recognize phosphorylated tyrosine amino acid residues. The peptide domains also have independent sites for the recognition of amino acid residues surrounding the phosphotyrosine residue(s).
When a receptor protein tyrosine kinase binds an extracellular ligand, receptor dimerization is induced, which, in turn, leads to intermolecular autophosphorylation of the dimerized kinases (Schlessinger, J. and Ullrich, A., 1992, Neuron 9: 383-391). Receptor phosphorylation, therefore, creates SH2-binding sites, to which an adaptor protein may bind.
SH2 domains represent recognition motifs for specific tyrosine-phosphorylated peptide sequences and are usually accompanied by another conserved domain of 50-75 amino acid residues, known as the SH3 domain. The current view is that SH3 domains function, in part, as protein-binding from the cell surface that act to link signals transmitted from the cell surface to downstream effector genes such as ras (Pawson, T. and Schlesinger, J., 1993 Current Biology, 3:434-442).
On the basis of their primary structures, it is possible to divide SH-2 containing proteins into two main classes: Type I and Type II. (Schlessinger, J., and Ullrich, A, 1992, Neuron 9:383-391). Type I defines SH-2 containing have distinct enzymatic activities, such as phospholipase activity, tyrosine kinase activity, and putative GDP-GTP exchange functions. Proteins of this class are thought to exert their enzymatic activities and transmit signals upon tyrosine phosphorylation or by interacting with neighboring target proteins.
Type II SH-2 containing proteins are adaptor proteins that are composed of virtually only SH-2 and SH-3 domains. Mammalian growth factor receptor-binding protein (GRB-2) is a 26 kilodalton member of the type II SH-2 containing proteins that has one SH-2 domain flanked by two SH-3 domains (Lowenstein et al., 1992, Cell 70:43-442). The GRB-2 adaptor protein binds to tyrosine-phosphorylated growth factor receptors through its SH-2 domain and to, inter alia, proline-rich regions of the son of sevenless (SOS) guanine nucleotide exchange factor through its SH-3 domains (Buday, L. and Downward, J., 1993, Cell 73:611-620; Egan, S. E. et al., 1993, Nature 363:45-51; Li, N. et al., 1993, Nature 363:85-87; Gale, N. W. et al., 1993, Nature 363:88-92; Rozakis-Adcock et al., 1993, Nature 363:83-85; Chardin, P. et al., 1993, Science 260:1338-1343; Oliver, J. P. et al., Cell 73:179-35 191; Simon, M. A. et al., 1993, Cell 73:169-177). Therefore, binding of GRB-2 to the receptor kinases, allows for the recruitment of SOS to the plasma membrane, where Ras, a guanine-nucleotide binding signaling protein, is located (Schlessinger, J., 1993, TIBS 18:273-275). As a result of the recruitment of SOS to the inner cell membrane by GRB-2 upon growth factor receptor tyrosine phosphorylation, the active GTP bound form of Ras accumulates for downstream signaling (Gibbs, J. B. et al., 1990, J. Biol. Chem. 265:20437-2044; Satoh, T. et al., 1990, Proc. Natl. Acad. Sci. USA 87:5993-5997; Li, B. -Q. et al., 1992, Science 256:1456-1459; Buday, L. and Downward, J., 1993, Mol. Cell. Biol. 13:1903-1910; Medema, R. H. et al., 1993, Mol. Cell. Biol. 13:155-162).
2.5 CELL PROLIFERATIVE DISORDERS
Growth factors and their receptors are crucial for normal cellular functions but can also act as oncogenes leading to cell transformation, oncogenesis, and cell proliferative disorders, including cancer. Activation of the oncogenic potential of normal cellular proteins may occur, e.g., by the uncoupling of the binding of the extracellular ligand to its receptor and the intracellular cascade of reactions, by alteration of the enzymatic activity of signaling proteins, or by inappropriate binding of signaling proteins to cellular components.
For example, it is known that the BCR-ABL oncoprotein is involved in the pathogenesis of leukemias, such as Philadelphia chromosome-positive human leukemia. BCR-ABL exhibits regulated tyrosine kinase activity that is not regulated by the binding of a ligand. It has recently been demonstrated (Pendergast, A. M. et al., 1993, Cell 75:175-185) that a tyrosine-phosphorylated region of the BCR-ABL binds the SH-2 domain of GRB-2, and that this interaction activates the Ras signaling pathway.
Thus, there are multiple events which occur along a signal transduction pathway which appear to be required for the ultimate appearance of a cell proliferative disorder such as the form of leukemia described above. One approach to the treatment of oncogenenic, cell proliferative disorders would be to attempt to xe2x80x9cshort circuitxe2x80x9d abnormal signal transduction events which contribute to the appearance of such disorders, by interfering with one or more of these requisite events.
The amelioration of abnormal signal transduction events leading to cell proliferative disorder symptoms may be accomplished by, e.g., targeting and directly inhibiting the interactions of proteins in the signal transduction pathway. For example, in instances wherein the signal transduction event of interest involves an adaptor protein/protein tyrosine kinase interaction, the inhibition of such interactions may lead to the amelioration of cell proliferative disorder symptoms. The utility of this approach has been demonstrated using expression of signaling incompetent proteins in cells. For example, cells expressing a mutant form of Bcr-Abl which lacks the tyrosine residue necessary for binding of the GRB-2 SH2 domain, and which is thus signaling incompetent, no longer exhibit a transformed phenotype (RER) (Pendergast et al., supra).
However, there are many signal transduction proteins that contain at least one SH2 domain, and therefore, compounds that are not specific for a particular SH2-containing protein will shut down signal transduction pathways indiscriminately. If these non-specific compounds were administered to a subject suffering from a cell proliferative disorder, they might be toxic to the subject or cause side effects associated with shutting down numerous signal transduction pathways. Therefore, it is desirable to have compounds that are specific for one type of interaction, e.g. the GRB-2 SH2-phosphotyrosine or the GRB-2 SH3-polyproline interaction. The specific interference of the binding of GRB-2 with either an activated tyrosine kinase or a downstream protein could result in blocking an abnormal signal transduction pathway at a fairly early stage without blocking other pathways that rely on the interaction of other SH2-containing proteins with phosphotyrosine.
2.6 DIABETES MELLITUS
Diabetes mellitus is a group of syndromes characterized by hyperglycemia, altered metabolism of lipids, carbohydrates, and proteins, and an increased risk of complications from vascular disease. There are two main types of diabetes mellitus: insulin-dependent diabetes mellitus (IDDM or Type I diabetes) and non-insulin-dependent diabetes mellitus (NIDDM or Type II diabetes). Insulin is a peptide hormone produced by the body that stimulates glucose uptake by cells, lipogenesis, and other general anabolic effects. Virtually all forms of diabetes mellitus are due to a decrease in the circulating concentration of insulin and a decrease in the response of peripheral tissues to insulin.
Insulin is responsible for maintaining glucose homeostasis in the body. When there is an excess of glucose in the body, or when tissues require fuel, insulin is released and binds to its protein tyrosine kinase receptor. The conformational change in the receptor resulting from insulin binding causes tyrosine phosphorylation of the insulin receptor intracellular tyrosine kinases. This in turn begins a signal transduction cascade that ultimately results in uptake of glucose by the cells. Depending on the type of cell, the glucose can be metabolized or stored as fat or glycogen for later use when needed, e.g., during starvation.
Insulin therapy is currently the most effective treatment of virtually all IDDM and many NIDDM patients. Human, porcine, bovine, or a mixture of porcine and bovine insulin are used in therapeutic preparations. Insulin cannot be administered orally because the protein is digested in the stomach. Rather, insulin must be administered intravenously, intramuscularly, or preferably, subcutaneously. Insulin injection differs from normal secretion of insulin in two major ways: the kinetics do not mimic the normal rapid rise and decline of insulin secretion in response to ingestion of nutrients, and the insulin diffuses into the peripheral circulation instead of being released into the portal circulation, thus eliminating the preferential effect of secreted insulin on hepatic metabolic processes. Insulin must be purified and supplied in a pharmaceutically acceptable carrier or diluent and is only stable for a few days. Thus, in addition to not ideally mimicking physiological insulin production, insulin therapy is also relatively expensive and inconvenient.
Diabetic patients suffer from a variety of disorders due to prolonged exposure of tissues to elevated concentrations of glucose, including premature atherosclerosis, intercapillary glomerulosclerosis, retinopathy, neuropathy and ulceration and gangrene of the extremities. Moreover, insulin therapy itself causes side effects, including hypoglycemia, insulin allergy and resistance, lipoatrophy at the site of insulin injection, lipohyperatrophy at sites of high insulin concentration, and insulin edema.
Because of the problems associated with insulin therapy, research effort has focused on finding alternative therapies for diabetes, and in particular the development of oral hypoglycemic agents. Oral hypoglycemic agents currently in use include the class of compounds known as the sulfonylureas, which act by stimulating insulin release from pancreatic cells, and the biguanides, which increase insulin action in peripheral tissues and reduce hepatic glucose output due to inhibition of gluconeogenesis. In addition, xcex1-glucosidase inhibitors such as acarbose, which reduce intestinal absorption of carbohydrates, are also administered orally in the treatment of diabetes. However, there are many side effects associated with these oral hypoglycemic agents, including nausea and vomiting, cholestatic jaundice, agranulocytosis, aplastic and hemolytic anemias, generalized hypersensitivity reactions, and dermatological reactions associated with sulfonylureas; diarrhea, abdominal discomfort, nausea, metallic taste and anorexia associated with biguanides; and malabsorption, flatulence, and abdominal bloating associated with xcex1-glucosidase inhibitors.
Thus, in view of the serious drawbacks associated with the current therapies for diabetes mellitus, there is a need in the art for an effective treatment for diabetes, which does not involve the inconvenience of insulin injection, or the side effects caused by existing oral hypoglycemic drugs used to treat diabetes. Therefore, there remains a need in the art for a method of controlling diabetes that is convenient, effective, inexpensive, and without major side effects.
The present invention relates to methods and compositions for inhibition of cell signal transduction associated with cell proliferative disorders. Specifically, the present invention relates to particular indolylquinone compounds, and methods for using such compounds. In a preferred embodiment, the compounds of the invention inhibit the interaction of protein tyrosine kinases with the GRB-2 adaptor protein, resulting in inhibition and suppression of tumor growth. Thus, the compounds of the present invention are useful in the treatment of cancers involving solid tumors, and in particular, the inhibition and reversal of tumor growth.
The compounds of the present invention are described by the formula I below: 
or a pharmaceutically acceptable salt thereof, wherein:
A is monocyclic aryl, bicyclic aryl or heteroaryl;
R1 and R2 are each independently Br, Cl, F, I, H, OH, or xe2x80x94OCOR, wherein R is lower alkyl, aryl or alkylaryl;
R1xe2x80x3 and R2xe2x80x3 are each independently H, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, arylalkyl or aryl; and
R3 to R6 and R8 to R12 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, C2-Cm alkynyl, alkenylcarboxy, monocyclic aryl, bicyclic aryl, heteroaryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12, preferably 2-7, and m is an integer from 3 to 12, preferably 3-7.
Preferred compounds of the present invention are described by the formula II, below: 
or a pharmaceutically acceptable salt thereof, wherein:
A1 and A2 are each individually carboxy, monocyclic aryl, bicyclic aryl or heteroaryl;
R1 and R2 are each independently Br, Cl, F, I, H, OH, or xe2x80x94OCOR, wherein R is lower alkyl, aryl or alkylaryl;
R1xe2x80x3 and R2xe2x80x3 are each independently H, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, arylalkyl or aryl; and
R3 to R6 and R8 to R11 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, C2-Cm alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12, preferably 2-7, and m is an integer from 3 to 12, preferably 3-7.
Preferred compounds of the present invention are compounds of formula I wherein A is: 
wherein R1 to R5 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, C2-Cm alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12, preferably 2-7, and m is an integer from 3 to 12, preferably 3-7; or 
wherein R1xe2x80x2xe2x80x3 to R7xe2x80x2xe2x80x3 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, C2-Cm alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12, preferably 2-7, and m is an integer from 3 to 12, preferably 3-7.
In preferred embodiments, R1xe2x80x2 to R5xe2x80x2 are H, and R1xe2x80x2xe2x80x3 to R7xe2x80x2xe2x80x3 are H.
Preferred compounds of the present invention also include compounds of formula II wherein A1 and A2 are each independently 
wherein R1xe2x80x2 to R5xe2x80x2 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, C2-Cm alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12, preferably 2-7, and m is an integer from 3 to 12, preferably 3-7; or 
wherein R1xe2x80x2xe2x80x3 to R7xe2x80x2xe2x80x3 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, C2-Cm alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12, preferably 2-7, and m is an integer from 3 to 12, preferably 3-7.
In preferred embodiments, R1xe2x80x2 to R5xe2x80x2 are H, and R1xe2x80x2xe2x80x3 to R7xe2x80x2xe2x80x3 are H.
In addition, the present invention encompasses a pharmaceutical composition comprising a compound of the formula I or formula II, or a pharmaceutically acceptable salt thereof, and methods for using a compound or pharmaceutical composition of the invention in an animal. Preferably, the animal is a mammal, and most preferably, a human. In particular, the present invention encompasses a method for ameliorating the symptoms of a cell proliferative disorder. In some embodiments, the cell proliferative disorder involves an interaction between GRB-2 and protein tyrosine kinase, comprising administering a therapeutically effective amount of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof. The present invention also encompasses a method for ameliorating the symptoms of a cell proliferative disorder, wherein the cell proliferative disorder involves an interaction between GRB-2 adaptor proteins and protein tyrosine kinases, comprising administering a pharmaceutical composition comprising a compound of formula I or formula II. The present invention is based, in part, on the inventors"" discovery that the disclosed compounds inhibit interactions of the GRB-2 adaptor protein with phosphorylated tyrosine kinases, thereby interrupting the cascade of cellular events which can lead to the development of cancer. Thus, the present invention also relates to methods for ameliorating symptoms of cell proliferative disorders associated with GRB-2 adaptor protein function, comprising administering an effective amount of a compound of formula I or formula II, or a pharmaceutical composition comprising a compound of formula I or formula II. The invention encompasses methods for treating a cell proliferative disorder. In certain embodiments, the cell proliferative disorder involves a protein tyrosine kinase/GRB-2 adaptor polypeptide complex. In some embodiments, the cell proliferative disorder involves an interaction between GRB-2 and tyrosine kinase.
The present invention also provides a method for ameliorating the symptoms of a cell proliferative disorder, comprising administering a therapeutically effective amount of a compound of the formula III below: 
or a pharmaceutically acceptable salt, wherein:
R1 and R2 are each independently Br, Cl, F, I, H, OH, or xe2x80x94OCOR, wherein R is lower alkyl, aryl or alkylaryl;
R1xe2x80x3 and R2xe2x80x3 are each independently H, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, arylalkyl or aryl; and
R3 to R12 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, C2-Cm alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12, preferably 2-7, and m is an integer from 3 to 12, preferably 3-7.
In addition, the present invention encompasses method of ameliorating the symptoms of a cell proliferative disorder, wherein the cell proliferative disorder involves an interaction between GRB-2 and a tyrosine kinase, which comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula III.
The present invention further comprises a method for ameliorating the symptoms of a cell proliferative disorder. In particular embodiments, the cell proliferative disorder involves an interaction between GRB-2 and tyrosine kinase, comprising administering a therapeutically effective amount of a compound of the formula (IV): 
or a pharmaceutically acceptable salt thereof, wherein:
R1, R2 and R30 are each independently Br, Cl, F, I, H, OH or xe2x80x94OCOR, wherein R is lower alkyl, aryl or alkylaryl;
R1xe2x80x3 is H, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, arylalkyl or aryl; and
R3 to R7 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12 and m is an integer from 3 to 12.
The present invention also encompasses a method of ameliorating the symptoms of a cell proliferative disorder comprising administering an effective amount of a pharmaceutical composition comprising a compound formula IV. The invention also relates to a method of inhibiting interactions between GRB-2 and tyrosine kinases, comprising administering an effective amount of a compound of formula III, a compound of formula IV, a pharmaceutical composition comprising a compound of formula III, or a pharmaceutical composition comprising a compound of formula IV.
In another aspect, the present invention relates to methods for treating insulin-related disorders, including, but not limited to diabetes, insulin resistance, insulin deficiency and insulin allergy, which comprise administering to a patient a therapeutically effective amount of a compound of formula I, formula II, formula III, or formula IV or a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula I, formula II, formula III, or formula IV. It has been discovered that the compounds of formulae II, ,III and IV have a hypoglycemic effect when administered to an animal, and are thus effective for treating the symptoms of insulin deficiency and insulin resistance in animals.
In particular, the present invention encompasses a method for treating diabetes or ameliorating the symptoms of diabetes comprising administering a therapeutically effective amount of a compound of formula I, II or III, or a pharmaceutically acceptable salt thereof. Administration of the compounds of formula I, II or III to a patient results in a lowering of the blood glucose level of the patient. Thus, the present invention encompasses a method of lowering the blood glucose level in an animal, comprising administering an effective amount of a compound of formula I, II or III, or a pharmaceutical composition comprising a compound of formula I, II or III. Without limiting the present invention to any particular mechanism of action to explain the hypoglycemic effect of the compounds of formulae I, II and III, it is believed that these compounds mimic the action of insulin in the body. In particular, it is believed that the compounds of the invention activate the insulin receptor tyrosine kinase in an animal, thereby triggering a cascade of cellular events leading to glucose uptake. Thus, the present invention also relates to a method of stimulating insulin receptor tyrosine kinase activity in an animal, comprising administering an effective amount of a compound of formula I, II or III.
The present invention encompasses methods for the treatment of both insulin-dependent or type I diabetes (formerly termed juvenile-onset of ketosis-prone diabetes) and non-insulin-dependent or type II diabetes (formerly termed adult-onset, maturity-onset or nonketotic diabetes). The methods of the present invention are suitable for treatment of mammals for veterinary use, or in humans for clinical uses. The invention relates to methods for treating and ameliorating the symptoms of insulin deficiency and other insulin disorders in an animal. The methods of the present invention are suitable for the treatment and amelioration of symptoms caused by a deficiency in insulin, or due to malfunctioning insulin-stimulated signal transduction leading to glucose uptake. In the case of insulin deficiency, the compounds described herein mimic the effects of insulin through interaction with insulin receptor kinase, thereby triggering the cascade of events resulting in glucose uptake and metabolism. Since the compounds of the invention stimulate and/or activate the insulin receptor protein tyrosine kinase, the methods of the invention are useful in the treatment of diabetic patients who do not produce enough insulin, and in diabetic patients who may produce insulin, but who are resistant to insulin.
In another aspect the present invention provides a method for the synthesis of indolylquinones which comprises reacting a substituted or unsubstituted 2,5-dihalo-1,4-benzoquinone with one or more substituted or unsubstituted indoles in a polar organic solvent and in the presence of metal carbonate.
In one embodiment, the present invention provides a method for preparing a symmetrical indolylquinone compound of the formula V: 
wherein:
R1 and R2 are each independently Br, Cl, F, I, H, OH or xe2x80x94OCOR, wherein R is, lower alkyl, aryl or alkylaryl;
R1xe2x80x3 is H, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, arylalkyl or aryl; and R3 to R7 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, C2-Cm alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12, preferably 2-7, and m is an integer from 3 to 12, preferably 3-7.
R1 and R2 are preferably Br, Cl, F, H or OH.
The method comprises reacting a substituted or unsubstituted 2,5-dihalo-1,4-benzoquinone, preferably a 2,5-dibromo-1,4-benzoquinone compound of the formula VI: 
wherein R1 and R2 are as defined above; with at least one indole of the formula VII: 
wherein
R1xe2x80x3 and R3-R7 are as defined above. The reaction is carried out in a polar organic solvent and in the presence of metal carbonate under mild conditions which are further discussed below.
The method may further comprise reacting the indolylquinone compound of formula V with an alkali metal hydroxide to produce a compound of the formula V, wherein R1 and R2 are OH.
Further, the method may further comprise reacting the indolylquinone compound of formula V wherein R1 and R2 are Br, with an alkali metal hydroxide and an alcohol of the formula Rxe2x80x2OH, wherein Rxe2x80x2 is lower alkyl or alkylaryl, to produce an indolylquinone compound of the formula V, wherein R1 is OR1xe2x80x2 and R2 is OR2xe2x80x2 wherein R1xe2x80x2 and R2xe2x80x2 are each independently lower alkyl or alkylaryl.
In another embodiment, the present invention provides a method for preparing an indolylquinone compound of the formula III, which comprises:
(a) reacting a substituted or unsubstituted 2,5-dibromo-1,4-benzoquinone compound of the formula VI: 
xe2x80x83wherein R1 and R2 are as defined above; with one equivalent of a first indole of the formula VII: 
wherein R1xe2x80x3 and R3-R7 are as defined above; in a polar organic solvent and in the presence of metal carbonate;
(b) reacting the intermediate product of step (a) with one equivalent of a second indole of the formula VIII: 
xe2x80x83wherein R2xe2x80x3 and R8-R12 are as defined above. Both reactions are carried out in a polar organic solvent and in the presence of metal carbonate under mild conditions which are further discussed below.
The invention also encompasses further reacting the indolylquinone compound of formula III with an alkali metal hydroxide to produce a compound of the formula III wherein R1 and R2 are OH.
Further, the invention encompasses reacting the indolylquinone of formula III wherein R1 and R2 are Br, F, Cl or I, with an alkali metal hydroxide and an alcohol of the formula Rxe2x80x2OH, wherein Rxe2x80x2 is lower alkyl or alkylaryl, to produce an indolylquinone compound of the formula III, wherein R1 is OR1xe2x80x2, and R2 is OR2xe2x80x2, wherein R1xe2x80x2 and R2xe2x80x2 are each independently lower alkyl, aryl or alkylaryl.
In another embodiment, the present invention provides a method for preparing a mono-indolylquinone compound of the formula IV: 
wherein:
R1, R2 and R30 are each independently Br, Cl, F, I, H, OH or xe2x80x94OCOR, wherein R is lower alkyl, aryl or alkylaryl;
R1xe2x80x3 is H, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, arylalkyl or aryl; and
R3 to R7 are each independently hydrogen, branched or unbranched C1-Cn alkyl, alkylcarboxy, C2-Cm alkenyl, alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy, hydroxyalkyl, C1-Cn alkoxy, nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from 2 to 12 and m is an integer from 3 to 12.
This method comprises reacting a substituted or unsubstituted 2,5-dibromo-1,4-benzoquinone compound of the formula VI: 
wherein R1 and R2 are as defined above, with one indole of the formula VII: 
wherein
R1xe2x80x3 and R3-R7 are as defined above. The reaction is carried out in a polar organic solvent and in the presence of metal carbonate.
The method of the present invention may further comprise reacting the indolylquinone compound of formula IV with an alkali metal hydroxide to produce a compound of the formula IV wherein R1 and R2 are OH.
The method may further comprise reacting the indolylquinone compound of formula IV wherein R1, R2 and R30 are Br, F, Cl or I, with a mixture of an alkali metal hydroxide and an alcohol of the formula Rxe2x80x2OH, wherein Rxe2x80x2 is lower alkyl or alkylaryl, to produce an indolylquinone compound of the formula IV wherein R1 is OR1xe2x80x2 and R2 is OR2xe2x80x2 wherein R1xe2x80x2 and R2xe2x80x2 are each independently lower alkyl, aryl or alkylaryl.
In another embodiment, the present invention further encompasses methods for producing large quantities of known, naturally occurring indolylquinones in high purity and in high yield. In yet another embodiment, the present invention is directed to known, synthetically prepared naturally occurring indolylquinones of high purity which are obtainable in large quantities and in high yield. The invention also encompasses the preparation of novel monoindolylquinones, i.e., compounds substituted with only one indole, and the monoindolylquinone compounds, as described below.
xe2x80x9cProtein tyrosine kinasexe2x80x9d will, herein, be abbreviated xe2x80x9cPTKxe2x80x9d. It is to be understood that xe2x80x9cPTKxe2x80x9d may refer to either a transmembrane, receptor-type protein tyrosine kinase or a cytoplasmic protein tyrosine kinase, unless otherwise indicated.
By the term xe2x80x9calkylxe2x80x9d as used herein is meant a straight or branched chain saturated hydrocarbon group having from 1 to 20 carbons, preferabably 1-12 carbons, such as methyl, ethyl, isopropyl, n-butyl, s-butyl, t-butyl, 3-methyl-n-butyl, n-amyl, isoamyl, n-hexyl, n-octyl and n-decyl; xe2x80x9calkenylxe2x80x9d and xe2x80x9calkynylxe2x80x9d are used to mean straight or branched chain hydrocarbon groups having from 2 to 12 carbons and unsaturated by a double or triple bond respectively, such as vinyl, allyl, propargyl, 1-methylvinyl, but-1-enyl, but-2-enyl, but-2-ynyl, 1 methylbut-2-enyl, pent-1-enyl, pent-3-enyl, 3-methylbut-1-ynyl, 1,1-dimethylallyl, hex-2-enyl and 1-methyl-1-ethylallyl; xe2x80x9calkylarylxe2x80x9d means the aforementioned alkyl groups substituted by a phenyl group such as benzyl, phenethyl, phenopropyl, 1-benzylethyl, phenobutyl and 2-benzylpropyl; xe2x80x9carylxe2x80x9d as used herein includes a monocyclic aromatic ring, including aromatic hydrocarbons; xe2x80x9cbicyclic arylxe2x80x9d as used herein includes bicyclic rings, wherein at least one ring is aromatic, including aromatic hydrocarbons; xe2x80x9cheteroarylxe2x80x9d as used herein includes monocyclic or bicyclic rings, wherein at least one ring is heteroaromatic, including heteroaromatic hydrocarbons; the term xe2x80x9chydroxy-alkylxe2x80x9d means the aforementioned alkyl groups substituted by a single hydroxyl group such as 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxybutyl, 1-hydroxybutyl and 6-hydroxyhexyl.
The term xe2x80x9csubstitutedxe2x80x9d as used herein means that the group in question may bear one or more substituents including but not limited to a radical in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s). Typical substituents include, but are not limited to, alkoxy, xe2x80x94X, xe2x80x94R, xe2x80x94Oxe2x88x92, xe2x95x90O, xe2x80x94OR, xe2x80x94Oxe2x80x94OR, xe2x80x94SR, xe2x80x94Sxe2x88x92, xe2x95x90S, xe2x80x94NRR, xe2x95x90NR, xe2x80x94CX3, xe2x80x94CN, xe2x80x94OCN, xe2x80x94SCN, xe2x80x94NCO, xe2x80x94NCS, xe2x80x94NHCHO, xe2x80x94NHCOC1-C4alkyl, xe2x80x94NHCOCH3, xe2x80x94NHCOCH2Cl, xe2x80x94NHCOCHCl2, xe2x80x94NHCOCCl3, xe2x80x94NHCOCF3, xe2x80x94NHCOCH2C6H4xe2x80x94oxe2x80x94NO2, xe2x80x94NHCOCH2OC6H4xe2x80x94oxe2x80x94NO2, xe2x80x94NHCOCH2COCH3, xe2x80x94NHCOCH2xe2x80x94N+C5H5Clxe2x88x92, xe2x80x94NHCOCH2NHCS2CH2C6H5, xe2x80x94NHCOCH2CH2C6H5, xe2x80x94NHCOCH2CH2C6H4xe2x80x94pxe2x80x94OH, xe2x80x94NHCOCH2CH2C6H4xe2x80x94oxe2x80x94NO2, xe2x80x94NHCOC(CH3)2OC6H4xe2x80x94oxe2x80x94NO2, xe2x80x94NHCOC(CH3)2OC6H4xe2x80x94oxe2x80x94Nxe2x95x90NC6H5, xe2x80x94NHCO(CH2)3Cl, xe2x80x94NHCOCH(CH3)2, xe2x80x94NHCOCHxe2x95x90CHC6H4xe2x80x94oxe2x80x94NO2, xe2x80x94NHCO-2-pyridyl, xe2x80x94NO, xe2x80x94NO2, xe2x95x90N2, xe2x80x94N3, xe2x80x94NHOH, xe2x80x94S(O)2Oxe2x88x92, xe2x80x94S(O)2OH, xe2x80x94S(O)2R, xe2x80x94P(O)(Oxe2x88x92)2, xe2x80x94P(O)(OH)2, xe2x80x94C(O)R, xe2x80x94C(O)X, xe2x80x94C(S)R, xe2x80x94C(S)X, xe2x80x94COOH, xe2x80x94C(O)OR, xe2x80x94C(O)Oxe2x88x92, xe2x80x94C(S)OR, xe2x80x94C(O)SR, xe2x80x94C(S)SR, xe2x80x94C(O)NRR, xe2x80x94C(S)NRR and xe2x80x94C(NR)NRR, where each X is independently a halogen (preferably xe2x80x94F, xe2x80x94Cl or xe2x80x94Br) and each R is independently xe2x80x94H, alkyl, lower alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, bicyclic aryl, hydroxy-alkyl and other substituents known to those skilled in the art.
Other features and advantages of the invention will be apparent from the following description of the p embodiments thereof, and from the claims.